Project Summary The 3D topology of the genome plays a critical role in transcriptional regulation in health, disease and development. While next generation sequencing techniques have revealed static views of global genome architecture, very little is known about the mechanisms that control dynamic topological rearrangements. We have established a system in budding yeast ? the Hsf1-mediated heat shock response ? in which concerted structural rearrangements within and between a set of specific target genes take place. During heat shock, Hsf1 drives its target genes dispersed on different chromosomes to undergo striking changes in conformation and coalesce into discrete, transcriptionally active foci. These Hsf1 target genes encode a dedicated group of protein homeostasis (proteostasis) factors, so-called ?Heat Shock Proteins? (HSPs). Through their regulation, the human orthologue of Hsf1 (hHSF1) has been suggested to be a clinical target in malignant cancers and in neurodegenerative diseases. Thus, this proposal has dual significance: it will both elucidate mechanisms that control chromatin conformational dynamics and reveal how Hsf1 coordinates expression of the proteostasis machinery. The Hsf1-mediated heat shock response in budding yeast represents an ideal model system to investigate the regulation and function of 3D genome rearrangements for two reasons: 1) The magnitude, rapidity and specificity of the intra- and intergenic rearrangements are all unprecedented and thus represent exciting, novel biology; and 2) It will allow us to leverage the power of yeast gene- tics to dissect the mechanisms and define the functional relevance of genome topology dynamics. Aim 1 will employ fluorescence microscopy in combination with pharmacological and genetic approaches to investigate the role of nuclear F-actin in driving coalescence of model Hsf1 target genes and regulating their transcription. Aim 2 will use powerful molecular techniques ? chromatin immunoprecipitation (ChIP), chromosome conformation capture (3C) and Reverse -Transcription qPCR (RT-qPCR) ? to investigate the role of filamentous actin, as well as its regulators and binding proteins, in orchestrating the 3D nuclear architecture of HSP genes and their transcriptional response to heat shock. In support of the NIH mission, the precedents established in this proposal will inform therapeutic efforts aimed broadly at 3D genome regulation and may suggest novel molecular handles with which to modulate Hsf1 and the proteostasis machinery to treat cancer and neurodegenerative diseases.